Fig. 2

Characterization of the maturation process of SorLA proteins in HEK293 cells. Western blot analyses after glycosidase treatments of wild-type full-length (SorLA^FL) (a, b) or secreted truncated (SorLA⁷⁵⁹ or SorLA²¹³¹) (c-g) forms of SorLA proteins transiently overexpressed in HEK293 cells. Blots were probed with an anti-SorLA antibody and representative blots are presented. Cellular lysates (intracellular) or supernatants (secreted) were treated in the absence or presence of the glycosidases PNGase F, Neuraminidase (Neu), O-Gycosidase (O-Glyc). Untreated samples (–) were incubated under the same conditions but without enzymes. PNGase F cleaves both high-mannose, hybrid and complex N-glycans from glycoproteins. Endo H cleaves preferentially high-mannose type N-glycans, but not complex N-glycans. Neuraminidase cleaves sialic acid from either complex or O-glycans. O-glycosidase removes O-linked glycans, but this cleavage is more effective only after removing terminal sialic residues by a neuraminidase. The positions of immature core-glycosylated and mature complex-glycosylated forms of SorLA are indicated with solid and empty arrowheads, respectively. Resulting corresponding species obtained after neuraminidase treatment are indicated with dashed arrowheads. The molecular masses of marker proteins in kDa are shown on the left